Involvement of NFƙB and MAPK signaling pathways in the preventive effects of Ganoderma lucidum on the inflammation of BV-2 microglial cells induced by LPS

Abstract

Ganoderma lucidum extract (GLE) is a potent ancient Asian remedy for the treatment of various diseases. This study investigated GLE preventive effects on LPS-stimulated inflammation of BV-2 microglial cells. The results show that pre-treatment with GLE decreased expression of pro-inflammatory cytokines: G-CSF, IL1-α, MCP-5, MIP3α, and, with a higher effect in MIP3α. In RT-PCR assays, pre-treatment with GLE decreased mRNA expression of CHUK, NFκB1/p150, and IKBKE (NFƙB signaling), which may be associated with the neuropathology of Alzheimer's disease. The data show GLE inhibiting ability on pro-inflammatory mediators' release and suggest a potential role of GLE in neurodegenerative disease prevention.

Keywords: Alzheimer's disease; And IKBKE; Ganoderma lucidum; Inflammatory cytokines; MIP3α; NFKB1/p50.

Fig. 1.

Dose-response decrease in cell viability by GLE in BV-2 microglia cells. GLE tested concentrations ranged from 0.5 to 1.3 mg/ml. All experiments were performed at least 3 times (n = 5) and kept at 5% CO₂ and 37 °C. The cytotoxic effect was measured after 24 h using Alamar Blue®. (A) Cells were treated with different concentrations of GLE. (B) Cells were pre-treated with different concentrations of GLE and stimulated with LPS (1 μg/ml) after 1 h. The data are presented as the mean ± S.E.M. Statistically significant differences between control vs. treatments were evaluated by a one-way ANOVA, followed by Dunnett’s multiple comparison tests. *p < .05, ***p < .001, ****p < .0001, ns = p > .05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2.

Dose-response decrease in nitric oxide production by GLE pre-treatment in BV-2 microglial cells stimulated by LPS. The concentrations of GLE ranged from 0.0125 to 0.6 mg/ml. All experiments were performed at least 3 times (n = 5) at 5% CO₂ and 37 °C for 24 h. Cells were pre-treated with GLE and after 1 h stimulated with LPS (1 μg/ml). The amount of nitrite was measured using Griess reagent. The data are presented as mean ± S.E.M Statistically significant differences between LPS vs. GLE + LPS-treatments were evaluated by a one-way ANOVA, followed by Dunnett’s multiple comparison tests. *p < .05, **p < .01, ns = p > .05.

Fig. 3.

Inhibition of cytokines expression by GLE pre-treatment in BV-2 microglial cells stimulated by LPS. A- Array layout used to assess chemokines/cytokines expression in the cell-free supernatants, highlighting the proteins downregulated by GLE and chemiluminescent spot intensity of supernatants derived from BV-2 cells showing cytokine changed expression after treatments. B- Graphs represent normalized protein expression of G-CSF, IL1α, MCP-5, MIP3α, and RANTES modulated by different treatments in BV-2 cells. Data are expressed as % of control arrays (mean ± S.E.M.) and correspond to normalized dot spot intensities from the cytokine arrays calculated based on the positive controls found in the corners of each one of the membranes using RAYBIO®ANALYSIS software. Blots and graphs represent the supernatants of: control (cells + dH₂O), GLE (0.5 mg/ml), LPS (1 μg/ml), and GLE (0.5 mg/ml) + LPS (1 μg/ml) after 1 h, in a 24-h treatment period (n = 3). Statistically significant differences between control vs. treatments (*), and LPS vs. GLE + LPS (^#) were evaluated by a one-way ANOVA, followed by Dunnett’s multiple comparison tests. *p < .05, **p < .01, ***p < .001, ^#p < .05, ^###p < .01, ns = p > .05.

Fig. 4.

Inhibition of protein expression of G-CSF, IL1α, MCP-5, MIP3α, and RANTES by GLE pre-treatment in BV-2 cells stimulated by LPS, using ELISA assay. The effect of the pre-treatment with GLE on G-CSF, IL1α, MCP-5, MIP3α, and RANTES protein expression in BV-2 cells stimulated by LPS was investigated with individual ELISAs. Each data point represents the mean ± S.E.M. of three independent experiments (n = 3), representing 4 treatments: control (cells + dH₂O), GLE (0.5 mg/ml) only, LPS (1 μg/ml) only, and GLE (0.5 mg/ml) + LPS (1 μg/ml) after 1 h, in a 24-h treatment period. Statistically significant differences between control vs treatments (*), and LPS vs GLE + LPS (^#) were evaluated by a one-way ANOVA, followed by Dunnett’s multiple comparison tests. **p < .01, ***p < .001, ****p < .0001, ^#p < .05, ^##p < .01, ^###p < .001, ^####p < .0001, ns = p > .05.

Fig. 5.

Down regulation of IRAK1, NOD1, CHUK, NFKB1/p50, and IKBKE mRNA expression by GLE pre-treatment in BV-2 microglial cells stimulated by LPS. Each data point represents the mean ± S.E.M. of three independent experiments (n = 3), representing 4 treatments: control (cells + dH₂O), GLE (0.5 mg/ml) only, LPS (1 μg/ml) only, and GLE (0.5 mg/ml) + LPS (1 μg/ml) after 1 h, in a 24-h treatment period. Statistically significant differences between control vs treatments (*), and LPS vs GLE + LPS (^#) were evaluated by a one-way ANOVA, followed by Dunnett’s multiple comparison tests. *p < .05, **p < .01, ***p < .001, ****p < .0001, ^##p < .01, ^###p < .001, ns = p > .05.